Irreversible tissue damage during myocardial infarction often leads to congestive heart failure. Implantation of exogenous cells in the heart, either as a cell suspension or tissue patch, is proposed for treatment of post-infarction disease. Although promising, initial clinical trials with injection of skeletal myoblasts and bone marrow derived stem cells have yielded only marginal improvements in cardiac function, thus prompting the quest for a better cell source. Novel, actively pursued cells are cardiogenic in nature and include adult resident cardiac stem cells (CSCs), embryonic stem cell derived cardiomyocytes (ESC-CMs) or cardiovascular progenitor cells (ESC-CPCs), and recently, cardiac progenitors isolated from induced pluripotent stem cells (iPSCs). Of the considered cell types, CSCs and ESC- or iPSC-derived cardiovascular progenitors can differentiate into cardiomyocytes, vascular smooth muscle, and endothelial cells and, at least in theory, may reconstitute both cardiac muscle tissue and its vasculature. Therefore, in this proof-of-concept study we propose to test the ability of mouse ESC-CPCs to form a functional cardiac tissue patch when placed in a 3-dimensional cardio-mimetic environment. Our main hypothesis is that, owing to the presence of supporting endothelial and smooth muscle cells, the ESC-CPC patches will exhibit superior structure and function compared to patches made of pure cardiomyocytes (ESC-CMs). Specific aims of this project are to: 1) develop a tissue culture bioreactor for electro-mechanical stimulation of engineered patches that mimics the contraction phases of the native cardiac cycle, 2) apply this cardio- mimetic stimulation to ESC-CPCs embedded in hydrogel/nanofiber biomaterials to create aligned and differentiated cardiac tissue patches, and 3) systematically assess electrical propagation and generated contractile forces in the obtained ESC-CPC patches in comparison to patches made of pure ESC-CMs. Towards the end of the project, the optimized design rules for engineering the ESC-derived cardiac tissue patch will be tested with mouse iPSC-CMs. In the future, this 3-dimensional cardiomimetic culture system will be applied as a reproducible test bed for studying the potential of different stem cells to undergo cardiogenesis in vitro. The obtained knowledge will be finally applied to constructing a functional cardiac tissue patch made of human cells and to testing its potential to restore cardiac function after infarction. PUBLIC HEALTH RELEVANCE: Heart failure is one of the most prominent cardiac diseases in USA that develops due to irreversible damage of heart tissue following a heart attack. This proposal describes a proof-of-concept study to engineer a living heart tissue substitute (cardiac patch) starting from novel cardiogenic stem cells. Engineered cardiac patch has a potential to be used in the future as a replacement for damaged heart tissue and to prevent the occurrence or progression of heart failure.